Type 1 diabetes is an autoimmune disease caused by T-lymphocyte generated ROS-mediated destruction of the insulin-producing beta cells. Mitochondrial and glycolytic enzyme dysfunction, pyridine cofactor and triosephosphate imbalance, and poly-ADP ribose polymerase (PARP) activation have each been implicated, suggesting that a metabolic mediator such as the NAD+/NADH ratio may be affected. We propose that zinc (Zn2+) potentiates some of these dysfunctions. Zn2+ is prevalent in the pancreas where it is released from beta-cell insulin secretory granules, both free and bound to insulin, and is taken back up into neighboring beta-cells. Zn2+ toxicity was recently implicated in islet-cell death that occurs secondarily to acute streptozotocin (STZ) exposure in vitro and in vivo. We show that niacinamide, pyruvate, NAD+, and sirtinol prevent Zn2+toxicity in neurons and beta-cells by counteracting the Zn2+-induced decrease in NAD+ levels in vitro (subset work in vivo). Niacinamide was shown to be an effective prophylactic for human, and animal type-1 diabetes if given before onset. Recently, we demonstrated that NOD animals have abnormally strong Zn2+ staining of islets as they age, and pyruvate attenuates beta-cell death, and diabetes in the NOD model. We propose that the autoimmune reaction stresses beta-cells which in turn release toxic concentrations of Zn2+. This causes death of adjoining beta-cells by NAD+depletion. Manipulations which raise [NAD+]i or lower [Zn2+]i will be protective. Niacinamide is an NAD+catabolism inhibitor and the precursor of NAD+, and the conversion of pyruvate to lactate acts to restore NAD+levels and metabolic enzymes, thereby reducing death. Sirtinol is a specific inhibitor of the sirtuin pathway which consists of NAD+dependent protein deacetylases. In specific aim 1, we will 1) measure [Zn2+]i, and 65Zn2+accumulation in isolated islets induced by Zn2+, cytokine, and streptozotocin exposures; 2) Determine the effects of these exposures on beta-cell metabolism, and the ability of pyruvate, niacinamide, and sirtinol to restore metabolism; 3) Lentiviral or genetic manipulation of NAD+ synthesis and catabolism will be used to explore the specific pathways involved in Zn2+ mediated NAD+ loss and beta-cell death. In specific aim 2, we will determine the ability of pyruvate, and niacinamide to reduce the metabolic derangements and diabetic incidence in the NOD model. Zinc transporter 5 (ZnT5) was suggested to load Zn2+ into secretory vesicles in beta-cells. SIRT-1 is the founding member of the sirtuin pathway which was implicated in neuronal and beta-cell Zn2+ toxicity, and Wlds overexpresses an NAD+synthetic enzyme. In aim 3, we will characterize ZnT5 -/-, +/+, Wlds, and SIRT-1-/-animals for diabetes incidence, beta-cell death, Zn2+ staining, and insulin release after MLDS streptozotocin-exposure. These experiments will test novel therapeutic compounds (pyruvate, sirtinol) and mechanisms (NAD+loss, sirtuins) involved in Zn2+ mediation of beta-cell death in type-1 diabetes. [unreadable] [unreadable] [unreadable]